原发性高血压儿童肠道菌群多样性分析

Analysis of gut microbiota diversity in children with primary hypertension

目的观察原发性高血压儿童肠道菌群分布情况,探讨肠道菌群多样性与血压的相关性。方法2022年5—8月首都儿科研究所附属儿童医院诊治原发性高血压患儿40例,根据血压分为高血压1级组15例和高血压2级组25例;同期选择血压正常儿童20例为对照组。收集3组儿童粪便样本,进行16S rDNA测序,采用Alpha多样性分析(Observed species指数、Goods coverage指数、Chao1指数、Shannon指数、Simpson指数、PD-whole tree指数)评估群落内物种多样性及丰富度,并绘制稀释曲线、Rank abundance曲线;采用Beta多样性分析(PCoA分析、Adonis分析、门水平分析)评估群落间物种多样性;采用LEfSe分析组间差异物种。结果高血压2级组、高血压1级组Chao1指数(182.869±90.187、159.286±64.650)均低于对照组(202.272±60.972)(P<0.05),高血压2级组高于高血压1级组(P<0.05);3组Observed species、Goods coverage、Shannon、Simpson、PD-whole tree指数比较差异均无统计学意义(P>0.05)。稀释曲线结果显示,各样本稀释曲线随序列数增加而趋于平缓,测序深度合理,群落内物种丰富度较高。Rank abundance曲线结果显示,高血压2级组物种丰富度及均匀度均高于高血压1级组、对照组,对照组高于高血压1级组。PCoA分析结果显示,高血压2级组与高血压1级组矩阵重叠度高,均与对照组矩阵存在明显距离(P=0.001,P=0.001)。Adonis分析结果显示,3组物种丰富度存在差异(F=1.263,P=0.029)。肠道菌群门水平上,3组均以拟杆菌门、厚壁菌门为主(约占90%);高血压1级组、对照组拟杆菌门占比相近,高血压2级组拟杆菌门占比降低;3组厚壁菌门占比相近。高血压2级组、高血压1级组变形菌门、放线菌门、热脱硫杆菌门、梭杆菌门、黏菌门、芽单胞菌门、纤维杆菌门占比均较对照组明显升高。LEfSe分析结果显示,高血压2级组丰度较高的菌群为乳酸杆菌、多尔氏菌、硒单胞子菌,高血压1级组丰度较高的菌群为苔藓杆菌、普氏梭杆菌,对照组丰度较高的菌群为普雷沃氏菌、毛螺菌、瘤胃球菌,3组间存在明显差异。结论原发性高血压儿童肠道菌群多样性及丰富度与正常血压儿童存在显著差异,随着血压增高,肠道菌群多样性及丰富度先降低后升高。

Objective To observe the distribution of gut microbiota in children with primary hypertension and to explore the correlation between gut microbiota diversity and blood pressure.Methods Forty children with primary hypertension were diagnosed and treated in Children's Hospital Affiliated to Capital Institute of Pediatrics from May 2022 to August2022,and were divided into grade 1 hypertension group(n=15)and grade 2 hypertension group(n=25)according to blood pressure.In the same period,another 20 children with normal blood pressure were selected as control group.The stool samples from three groups were collected,and were sequenced using 16S rDNA sequencing technology.Alpha-diversity analysis(Observed species index,Goods coverage index,Chao1 index,Shannon index,Simpson index and PD-whole tree index)was done to evaluate species diversity and richness within communities.The dilution curve and Rank abundance curve were drawn.Beta-diversity analysis(PCoA analysis,Adonis analysis,and phylum-level analysis)was done to assess species diversity among communities.LEfSe method was used to analyze the different species between groups.Results The Chao1 index was lower in grade 2 hypertension group(182.869±90.187)and grade 1 hypertension group(159.286±64.650)than that in control group(202.272±60.972)(P<0.05),and was higher in grade 2hypertension group than that in grade 1 hypertension group(P<0.05).The alpha-diversity indexes(Observed species,Goods coverage,Shannon,Simpson and PD-whole tree)were not different among three groups(P>0.05).The dilution curve results showed that the dilution curve of each sample tended to be gentle with the increase of sequence number,the sequencing depth was reasonable,and the species richness in the community was high.The Rank abundance curve showed that the species richness and evenness were higher in grade 2 hypertension group than those in grade 1 hypertension group and control group,and higher in control group than those in grade 1 hypertension group.The PCoA analysis results showed that the matrix had a high overlap in grade 2 hypertension group and grade 1 hypertension group,which showed a significant distance compared with control group(P=0.001,P=0.001).The Adonis analysis results showed that there were differences in species richness among three groups(F=1.263,P=0.029).At the phylum level,the Bacteroidetes and Firmicutes were dominant in three groups(about 90%).The proportion of Bacteroidetes was similar in grade 1hypertension group and control group,and decreased in grade 2 hypertension group.The proportion of Firmicutes was similar in three groups.The proportions of Proteobacteria,Actinomycetes,Thermodesulphuricobacteria,Clostridium,Myxomycetes,Blastomonas and Fibrinobacteria were significantly higher in grade 2 and 1 hypertension groups than those in control group.LEfSe analysis results showed that the bacteria with higher abundance were Lactobacillus,Doelsiella and Selenomonas in grade 2 hypertension group,were Bryobacterium and Clostridium prevotella in grade 1 hypertension group,and were Prevotella,Spirillum pilosa and Ruminococcus in control group;which showed significant differences among three groups.Conclusion The diversity and richness of gut microbiota in children with primary hypertension are significantly different from those in children with normal blood pressure,and they show a trend of decrease and subsquent increase with the elevation of blood pressure.